Perry S F, Furimsky M, Bayaa M, Georgalis T, Shahsavarani A, Nickerson J G, Moon T W
Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5.
Biochim Biophys Acta. 2003 Dec 30;1618(2):175-84. doi: 10.1016/j.bbamem.2003.09.015.
Using degenerate primers, followed by 3' and 5' RACE and "long" PCR, a continuous 4050-bp cDNA was obtained and sequenced from rainbow trout (Oncorhynchus mykiss) gill. The cDNA included an open reading frame encoding a deduced protein of 1088 amino acids. A BLAST search of the GenBank protein database demonstrated that the trout gene shared high sequence similarity with several vertebrate Na(+)/HCO(3)(-) cotransporters (NBCs) and in particular, NBC1. Protein alignment revealed that the trout NBC is >80% identical to vertebrate NBC1s and phylogenetic analysis provided additional evidence that the trout NBC is indeed a homolog of NBC1. Using the same degenerate primers, a partial cDNA (404 bp) for NBC was obtained from eel (Anguilla rostrata) kidney. Analysis of the tissue distribution of trout NBC, as determined by Northern blot analysis and real-time PCR, indicated high transcript levels in several absorptive/secretory epithelia including gill, kidney and intestine and significant levels in liver. NBC mRNA was undetectable in eel gill by real-time PCR. In trout, the levels of gill NBC1 mRNA were increased markedly during respiratory acidosis induced by exposure to hypercarbia; this response was accompanied by a transient increase in branchial V-type H(+)-ATPase mRNA levels. Assuming that the branchial NBC1 is localised to basolateral membranes of gill cells and operates in the influx mode (HCO(3)(-) and Na(+) entry into the cell), it would appear that in trout, the expression of branchial NBC1 is transcriptionally regulated to match the requirements of gill pHi regulation rather than to match trans-epithelial HCO(3)(-) efflux requirements for systemic acid-base balance. By analogy with mammalian systems, NBC1 in the kidney probably plays a role in the tubular reabsorption of both Na(+) and HCO(3)(-). During periods of respiratory acidosis, levels of renal NBC1 mRNA increased (after a transient reduction) in both trout and eel, presumably to increase HCO(3)(-) reabsorption. This strategy, when coupled with increased urinary acidification associated with increased vacuolar H(+)-ATPase activity, ensures that HCO(3)(-) levels accumulate in the body fluids to restore pH.
利用简并引物，随后进行3'和5' RACE以及“长”PCR，从虹鳟（Oncorhynchus mykiss）鳃中获得了一个连续的4050 bp cDNA并进行了测序。该cDNA包含一个开放阅读框，编码一个推导的1088个氨基酸的蛋白质。对GenBank蛋白质数据库进行BLAST搜索表明，鳟鱼基因与几种脊椎动物的Na(+)/HCO(3)(-)共转运体（NBCs），特别是NBC1，具有高度的序列相似性。蛋白质比对显示，鳟鱼NBC与脊椎动物NBC1的同一性大于80%，系统发育分析提供了额外的证据，表明鳟鱼NBC确实是NBC1的同源物。使用相同的简并引物，从鳗鱼（Anguilla rostrata）肾脏中获得了NBC的部分cDNA（404 bp）。通过Northern印迹分析和实时PCR确定的鳟鱼NBC组织分布分析表明，在包括鳃、肾脏和肠道在内的几种吸收/分泌上皮中，转录水平较高，在肝脏中也有显著水平。通过实时PCR在鳗鱼鳃中未检测到NBC mRNA。在鳟鱼中，暴露于高碳酸血症诱导的呼吸性酸中毒期间，鳃NBC1 mRNA水平显著增加；这种反应伴随着鳃V型H(+)-ATPase mRNA水平的短暂增加。假设鳃NBC1定位于鳃细胞的基底外侧膜并以流入模式运作（HCO(3)(-)和Na(+)进入细胞），那么在鳟鱼中，鳃NBC1的表达似乎是转录调控的，以匹配鳃细胞内pH值调节的需求，而不是匹配全身酸碱平衡的跨上皮HCO(3)(-)流出需求。与哺乳动物系统类似，肾脏中的NBC1可能在肾小管对Na(+)和HCO(3)(-)的重吸收中起作用。在呼吸性酸中毒期间，鳟鱼和鳗鱼肾脏中的NBC1 mRNA水平在短暂降低后均升高，推测是为了增加HCO(3)(-)的重吸收。这种策略与液泡H(+)-ATPase活性增加相关的尿酸化增加相结合，确保HCO(3)(-)水平在体液中积累以恢复pH值。
